Pharmaceutical chimeric receptor composition and method thereof

ABSTRACT

Disclosed herein provides a pharmaceutical composition and a disease therapy method. The pharmaceutical composition relates to an artificial chimeric antigen receptor (CAR). Specifically, the pharmaceutical composition includes a CAR protein that is highly specific to CD19 antigen, a vector that is capable of inducing a cell to generate the certain CAR 19 protein and a population of a modified mammal cell including the CAR19 protein, the vector or combination thereof. Furthermore, the artificial CAR19 includes a CD19 antigen-binding fragment, a transmembrane domain, and a signaling domain. The CD19 antigen-binding fragment is a single-chain variable fragment (scFv) having specific amino acid sequences. Additionally, the method relates to a cancer therapy by using said modified mammal cells. Furthermore, the method includes the steps of purifying a population of autologous cells, modifying the population of autologous cells with an artificial CAR, and administrating the modified autologous cells.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to PCT ApplicationPCT/CN2018/099502 filed on Aug. 9, 2018, which claims priority to U.S.Provisional Application Ser. No. 62/610,156 filed on, Dec. 23, 2017, andthe entire content of which is incorporated by reference to thisapplication.

FIELD

The present disclosure relates to pharmaceutical chimeric receptorcompositions. More particularly, it relates to pharmaceutical chimericantigen receptor 19 compositions and therapy methods using suchcomposition.

BACKGROUND

Cancer is a group of diseases characterized by the uncontrolled growthand spread of abnormal cells. If the growth and spread of cancer cellscannot be controlled, the host will die. Research shows that cancer ispredominately caused by external factors such as tobacco, infectiousorganisms, and an unhealthy diet; and internal factors such as inheritedgenetic mutations, hormones, and immune conditions. These factors mayact together or in sequence to cause cancer. Years may pass between theexposure to cancer causing factors and cancer detection. Cancertherapies may include surgery, radiation, chemotherapy, hormone therapy,immune therapy, and targeted therapy (drugs that interfere specificallywith cancer cell growth).

Leukemia can be classified into four types: Acute Myeloblastic Leukemia(AML), Chronic Myeloblastic Leukemia (CIVIL), Acute LymphoblasticLeukemia (ALL) and Chronic Lymphoblastic Leukemia (CLL). The majordifferences between the four types of leukemia are their progressionrate and the location in which the cancer develops. According to thereport from the Childhood Cancer Foundation, leukemia affects about 31%of the children with cancer, which is the highest of all cancer.Moreover, 95% of leukemia detected is acute leukemia.

Treatment strategies are different depending on the types leukemia(e.g., AML, CIVIL, ALL and CLL). Treatments may include chemotherapy,target therapy, radiation therapy, hematopoietic stem celltransplantation and/or supportive treatment. Because it is difficult todetermine whether all leukemia cells in the host have been killed,recurrence of leukemain happens often. Also cancer cells may developtolerance to regular chemotherapy.

The strategies for treating leukemia include chemotherapy, targetedtherapy, radiotherapy, stem cells transplantation, support therapy, etc.Due to cancer recurrence and cells developing tolerance, immunoetherapyhas recently become a popular alternative to conventional chemotherapy.One form of immunotherapy is to genetically modify T cells to targetantigens expressed on tumor cells through the expression of chimericantigen receptors (CARs). CARs are artificial designed antigen receptorsthat expressed on a human leucocyte through a genetic engineering torecognize tumor cell surface antigens. In 2011, Dr. Carl Junesuccessfully used modified T cells carrying CARs on the cell surface totreat leukemia patients. Porter et al., N Engl J Med, 2011; 365:725-733.

According to recent studies, tumor-specific antigens are not yet welldefined in most tumors. However, it is well known that CD19 is aneffective tumor target in B cell malignancies. Expression of CD19 isrestricted to normal and malignant B cells (Uckun, et al. Blood, 1988,71:13-29). Also, some studies demonstrated that it is effective toeliminate B lymphoma through targeting CD19. See for example, Salem etal., Indian J Hematol Blood Transfus, 2012, 28(2):89-96; Schewe et al.,Blood, 2017-01-764316; and Seidel et al., Mol Ther., 2016,24(9):1634-43.

Therefore, there is a need to develop a pharmaceutical CAR compositioncontaining small protein with highly specific recognition to CD19, andan effective method to treat patient with abundant CD19 expression byusing such pharmaceutical CAR composition.

SUMMARY

The present disclosure provides an artificial CAR protein including aCD19 antigen-binding fragment, a transmembrane domain, and a signalingdomain. Further, the CD19 antigen-binding fragment is a single-chainvariable fragment (scFv) including a heavy chain variable domain, alight chain variable domain and at least one linker between the heavyand the light chain variable domains. The heavy chain variable domainincludes a first amino acid sequence selected from SEQ ID NO: 8, SEQ IDNO: 10, SEQ ID NO: 12 or any combination thereof, and the light chainvariable domain includes a second amino acid sequence selected from SEQID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or any combination thereof. Thetransmembrane domain includes a transmembrane domain of CD28, IgG1, CD4,CD8α or any combination thereof. The signaling domain includes at leastone immunoreceptor tyrosine-based activation motif (ITAM), at least oneco-stimulatory molecule (CM) or the combination thereof.

In some embodiments of the present disclosure, the transmembrane domainof the artificial CAR protein is the transmembrane domain of CD8a.

In some embodiments of the present disclosure, the artificial CARprotein includes at least two CMs.

In some embodiments of the present disclosure, the at least one ITAM ofthe artificial CAR protein includes the CD3 zeta.

In some embodiments of the present disclosure, the at least one CM ofthe artificial CAR protein includes a CM of CD27, CD28, 4-1BB/CD137,OX40 herpesvirus entry mediator (HVEM) or any combination thereof.

In some embodiments of the present disclosure, the at least one CM ofthe artificial CAR protein includes the CM of 4-1BB/CD137.

The present disclosure also provides an expression vector of artificialCAR having a CD19 antigen-binding fragment sequence, a transmembranedomain sequence and a signaling domain sequence. Further, the CD19antigen-binding fragment sequence includes a heavy chain variable domainnucleic acid sequence, a light chain variable domain nucleic acidsequence and at least one linker between the heavy and the light chainvariable domain nucleic acid sequences. The nucleic acid sequence of theheavy chain variable domain includes SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6 or any combination thereof, and the nucleic acid sequence of thelight chain variable domain includes SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5 or any combination thereof. The transmembrane domain sequenceincludes a transmembrane sequence of CD28, IgG1, CD4, CD8α or anycombination thereof. The signaling domain sequence includes at least oneITAM sequence, at least one CM sequence or the combination thereof.

The present disclosure further provides a pharmaceutical compositionhaving a population of a modified cell. Specifically, the modified cellincludes at least one artificial CAR protein, at least one artificialCAR expression vector or the combination thereof, both of which havingthe specific property as the previous description respectively.

In some embodiments of the present disclosure, the modified cell of thepharmaceutical composition is a mammalian cell.

In some embodiments of the present disclosure, the modified cell of thepharmaceutical composition is a lymphocyte.

In some embodiments of the present disclosure, the modified cell of thepharmaceutical composition is a T cell or a NK cell.

The present disclosure furthermore provides a method for treating amammal having a disease, disorder or a condition associated with anelevated expression of a CD19 antigen. Specifically, the method includesthe following steps: step (a) isolating peripheral blood from at leastone mammal; step (b) purifying a plurality of lymphocytes from theperipheral blood; step (c) generating a pharmaceutical compositionhaving at least one artificial CAR protein, at least one artificial CARexpression vector or the combination thereof using the lymphocyte; step(d) treating the mammalian recipient with at least one ofchemotherapeutic agent; and step (e) administrating the pharmaceuticalcomposition to the mammalian recipient.

In some embodiments of the present disclosure, the step (c) furtherincludes a step of amplifying the pharmaceutical composition.

In some embodiments of the present disclosure, the pharmaceuticalcomposition is autologous to at least one of the mammalian donor and atleast one of the mammalian recipient.

In some embodiments of the present disclosure, the pharmaceuticalcomposition is allogenic to at least one of the mammalian donor and atleast one of the mammalian recipient.

In some embodiments of the present disclosure, the lymphocyte isolatedfrom the peripheral blood is a T cell or a NK cell.

BRIEF DESCRIPTION OF THE DRAWINGS

One or more embodiments are illustrated by way of example, and not bylimitation, in the figures of the accompanying drawings, whereinelements having the same reference numeral designations represent likeelements throughout. The drawings are not to scale, unless otherwisedisclosed.

FIGS. 1A-1B are images of the schematic representations of thegene-transfer vector and transgene and the purity of PBMCs andlymphocytes purification. FIG. 1A illustrates the lentiviral vectors andtransgene that show the major functional elements. A lentiviral vector(pLAS5w.PeGFP-I2-Puro) directing expression of anti-CD19 seFv derivedfrom murine or human monoclonal antibody, human CD8α hinge andtransmembrane domain, as well as human 4-1BB/CD137 and CD3zeta signalingdomains, were produced. Constitutive expression of the transgene wasdirected by the inclusion of an EF-1α (elongation factor-1a promoter);LTR, long terminal repeat; RRE, rev response element; IRES, internalribosome entry site; eGFP, enhanced green fluorescence protein; andWPRE, woodchunk hepatitis virus post transcriptional regulatory element.FIG. 1B illustrates CD3 and CD56 expression of purified PBMCs andlymphocytes by surface staining. Further, the upward shift (on Y-axis)in the FACS staining pattern is due to the CD3 expression on thepurified cells, and the right shift (on X-axis) in the FACS stainingpattern is due to the CD56 expression on cells.

FIGS. 2A-2C are images of the schematic representations of the highexpression efficiency and the high expansion rate of UW022-CAR,UW024-CAR or UW026-CAR modified CD3⁺ lymphocytes. FIGS. 2A and 2B depictFab and GFP expression on the modified CD3⁺ lymphocytes after theconstruction by surface staining. Furthermore, as FIG. 2A shows, theupward shift in the FACS staining pattern is due to the Fab expressionon the purified cells, and the right shift in the FACS pattern is due tolentivirial vector incorporation of the modified CD3⁺ lymphocytes. FIG.2B depicts statistic data of the expression of Fab or GFP on themodified CD3⁺ lymphocytes after construction. Each data bar representsthe average of triplicate measurement of the Fab expression or the GFPexpression on each modified lymphocytes. FIG. 2C depicts the statisticdata of the cell number fold of various modified CD3⁺ lymphocytes indifferent time points after construction. Each data point represents theaverage of triplicate measurement of the modified lymphocytes.

FIGS. 3A-3F are images of the schematic representations of thecytotoxicity efficiency of the CAR19 modified CD3⁺ lymphocytes to twoCD19⁺ lymphoma cells (RS4;11 and Raji). FIGS. 3A and 3B depict theremaining quantity of RS4;11 or Raji cells after co-culturing with thedifferent modified CD3⁺ lymphocytes for 24 hours by surface staining.Furthermore, as FIGS. 3A and 3B show, the right shift of gating area inthe FACS staining pattern is due to the CD19 positive expression onRS4;11 or Raji cells. FIGS. 3C and 3D depict statistic data of theremaining quantity of RS4;11 or Raji cells after co-culturing with thedifferent modified CD3⁺ lymphocytes. Each bar represents the average oftriplicate measurement of the CD19⁺ cells. FIGS. 3E and 3F depictinterferon-γ (INF-γ) secretion ability of the different modified CD3⁺lymphocytes to RS4;11 or Raji cells after co-culture for 24 hours byenzyme-linked immunosorbent assay. Each bar represents the average oftriplicate measurement of the quantity of INF-γ.

FIGS. 4A-4B are images of the schematic representations where theallogeneic cytotoxicity of the CAR19 modified CD3⁺ lymphocytes to CD19⁺lymphoma cells (RS4;11 and Raji) is corresponding to the cell numbers ofthe modified CD3⁺ lymphocytes. FIG. 4A depicts the cytotoxic quantity ofthe different modified CD3⁺ lymphocytes in response to a different ratio(E:T, CAR19 T cells versus target tumor cells) of allogeneic lymphomacells (CD19⁺ cells: RS4;11 and Raji; and CD19⁻ cells K562) after 4 hoursco-culture. Each data point represents the average of triplicatemeasurement of the modified lymphocytes. FIG. 4B shows that the UW022transfected CD3+ lymphocytes release the most interferon-γ (INF-γ) afterco-culturing with the RS4;11 or Raji cells for 4 hours.

FIGS. 5A-5C is a series of images of the schematic representations of anin vivo cytotoxicity experiment design and in vivo cytotoxicityefficiencies of different modified CD3⁺ lymphocytes. FIG. 5A depicts thein vivo experiment design. NOD/SCID mice were given Raji/Luc⁺ cells atDay 0 and different modified CD3⁺ lymphocytes at Day 7. After, theNOD/SCID mice are imaged by the IVIS system every seven days. FIG. 5Bdepicts the tumor (Raji/Luc⁺ cells) growth at the respective time pointby an in vivo imaging system. FIG. 5C depicts statistic data of thebioluminescence intensity (BLI) of the tumor size monitored by the invivo imaging system. Each data bar represents the average of fiverepeated measurement of the BLI value.

The drawings are only schematic and are non-limiting. In the drawings,the size of some of the elements may be exaggerated and not drawn onscale for illustrative purposes. The dimensions and the relativedimensions do not necessarily correspond to actual reductions topractice of the invention. Any reference signs in the claims shall notbe construed as limiting the scope. Like reference symbols in thevarious drawings indicate like elements.

DETAILED DESCRIPTION OF THE DISCLOSURE

The making and using of the embodiments of the disclosure are discussedin detail below. It should be appreciated, however, that the embodimentsprovide many applicable inventive concepts that can be embodied in awide variety of specific contexts. The specific embodiments discussedare merely illustrative of specific ways to make and use theembodiments, and do not limit the scope of the disclosure.

The present disclosure relates to compositions and a method for treatingdisorders or diseases, such as cancer including but not limited tohematologic malignancies and solid tumors. The present disclosurerelates to a strategy of constructing an artificial CAR protein, anexpression vector of the artificial CAR and a pharmaceutical compositionthat may include molecules thereof that combine antibody-basedspecificity for a desired antigen (e.g., tumor antigen) with a T cellreceptor-activating intracellular domain to generate a chimeric proteinthat exhibits a specific anti-tumor cellular immune activity.

The present disclosure generally relates to a protein construction ofgenetically modified CAR. Moreover, the artificial CAR of the presentdisclosure is highly specific to the CD19 antigen. Therefore, theartificial CAR is referred to herein as CAR19 or anti-CD19 CAR.

The artificial CAR19 protein of the present composition generallyincludes three major domains located in an extracellular area, a cellmembrane area and an intracellular area respectively. The three majordomains include a CD19 antigen-binding fragment, a transmembrane domainand a signaling domain. Further, the CD19 antigen-binding fragment is ascFv having a heavy chain variable domain and a light chain variabledomain as well as a linker to connect the variable domain of heavy chainand light chain. Additionally, the heavy chain variable domain includesa first amino acid sequence, which is selected from SEQ ID NO: 8, SEQ IDNO: 10, SEQ ID NO: 12 or any combination thereof. The light chainvariable domain may include a second amino acid sequence, which isselected from SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 or anycombination thereof. In another aspect, the transmembrane domainincludes a transmembrane domain of CD28, IgG1, CD4, CD8α or anycombination thereof. In the other aspect, the signaling domain includesat least one ITAM, at least one CM or the combination thereof.

In some embodiments, the artificial CAR19 protein of the presentcomposition consists of the CD19 antigen-binding fragment, thetransmembrane domain and the signaling domain. That is, the CAR19protein only includes these three domains without any other domain.Moreover, the scFv also only includes one heavy chain variable domain,one light chain variable domain and one linker therebetween. Similarly,the first amino acid sequence of the heavy chain variable domain isselected from SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12. The secondamino acid sequence of the light chain variable domain is selected fromSEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11. In another aspect, thetransmembrane domain only includes a transmembrane domain of the CD28,IgG1, CD4 or CD8a. In the other aspect, the signaling domain onlyincludes an ITAM or a CM.

In some embodiments, the scFv of the CD19 antigen-binding fragmentfurther includes a linker sequence selected from SEQ ID NO: 13, SEQ IDNO: 14, SEQ ID NO: 15 or SEQ ID NO: 16. An another embodiments, theartificial CAR19 protein further includes at least one linker betweentwo protein domains others than the CD19 antigen-binding fragment, andthe linker includes a sequence selected from SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15 or SEQ ID NO: 16.

In some embodiments, the artificial CAR19 protein includes at least twoCD19 antigen-binding fragments. That is, there are more than one scFvmolecule. The first amino acid sequence of the heavy chain variabledomain may be a repeated sequence or any combination sequence selectedform SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11. Also, the second aminoacid sequence of the light chain variable domain may be a repeatedsequence of or any combination sequence of SEQ ID NO: 7, SEQ ID NO: 9 orSEQ ID NO: 11.

In some embodiments, the scFv of the artificial CAR19 proteincomposition includes a specific combination of the first amino acidsequence and the second amino acid sequence, which is the SEQ ID NO: 8with SEQ ID NO: 7, the SEQ ID NO: 10 with SEQ ID NO: 9, or the SEQ IDNO: 12 with SEQ ID NO: 11.

In some preferable embodiments, the artificial CAR19 protein includesthe first amino acid sequence of the heavy chain variable domain that isthe SEQ ID NO: 8, and the second amino acid sequence of the light chainvariable domain that is the SEQ ID NO: 7.

In some preferable embodiments, the transmembrane domain of theartificial CAR19 protein is the transmembrane domain of CD8a.

In some embodiments, the signaling domain of the artificial CAR19protein includes both the ITAM and the CM. In another embodiment, thecombination of the signaling domain of the artificial CAR19 proteinincludes at least one ITAM and at least two CM.

In some embodiments, the at least one ITAM of the signaling domain inthe artificial CAR19 protein is a CD3 zeta chain.

In some embodiments, the CM of the signaling domain in the artificialCAR19 protein includes a CM region of CD27, CD28, CD30, 4-1BB/CD137,OX40 or HVEM. In another embodiment, the number of the CM of thesignaling domain is at least two, therefore, the CMs include anycombination of the CM regions of CD27, CD28, CD30, 4-1BB/CD137, OX40 orHVEM. In one preferable embodiment, the artificial CAR19 proteinincludes one CM in the signaling domain, and the CM is 4-1BB/CD137 orCD28.

The present disclosure also relates to an expression vector that mayexpress a desired artificial CAR19 stably. A backbone of the artificialCAR19 expression vector generally is selected from the expression vectorsystem used in the mammalian system. Preferably, the expression vectoris a lentiviral vector. Further, the artificial CAR19 expression vectorincludes a CD19 antigen-binding fragment sequence, a transmembranedomain sequence and a signaling domain sequence. In one aspect, the CD19antigen-binding fragment sequence includes a nucleic acid sequence of aheavy chain variable domain, a nucleic acid sequence of a light chainvariable domain and a linker. The nucleic acid sequence of heavy chainvariable domain includes SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or anycombination thereof, and the nucleic acid sequence of light chainvariable domain includes SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or anycombination thereof. In another aspect, the transmembrane domainsequence includes a transmembrane sequence of CD28, IgG1, CD4, CD8α orany combination thereof. In the other aspect, the signaling domainsequence comprises an ITAM sequence, a CM sequence or the combinationthereof.

In some embodiments, the artificial CAR19 expression vector consists anucleic acid sequence of a vector backbone, a CD19 antigen-bindingfragment, a transmembrane domain and a signaling domain. That is, theCAR19 expression vector only includes these four nucleic acid sequenceswithout any other nucleic acid sequence. Further, the CD19antigen-binding fragment sequence is consisted of three nucleic acidsequences. That is, the CD19 antigen-binding fragment sequence onlyincludes the sequences of the heavy chain variable domain, the lightchain variable domain and the linker. The transmembrane domain sequenceonly consists of one sequence, which is the transmembrane sequence ofCD28, IgG1, CD4 or CD8a. The signaling domain sequence also onlyconsists of a single sequence, which is the CM sequence or the ITAMsequence.

In some embodiments, the artificial CAR19 expression vector includesmore than one (i.e., at least two) nucleic acid sequence of the heavychain variable domain or the light chain variable domain. In anotherword, the nucleic acid sequence of the heavy chain variable domain maybe a repeat of or any combination of SEQ ID NO: 2, SEQ ID NO: 4 or SEQID NO: 6, and the nucleic acid sequence of the light chain variabledomain may be a repeat of or any combination of SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO: 5.

In some embodiments, the artificial CAR19 expression vector includes aspecific nucleic acid sequence combination of the heavy chain variabledomain and the light chain variable domain, and the combination is SEQID NO: 2 with SEQ ID NO: 1, SEQ ID NO: 4 with SEQ ID NO: 3, or SEQ IDNO: 6 with SEQ ID NO: 5.

In one preferable embodiment, a nucleic acid sequence of the heavy chainvariable domain and the light chain variable domain of the artificialCAR19 expression vector are SEQ ID NO: 2 and SEQ ID NO: 1, respectively.

In some embodiments, a transmembrane domain sequence of the artificialCAR19 expression vector includes at least two transmembrane sequences.Therefore, the transmembrane domain sequence may be a repeated sequenceof or any combination sequence of the transmembrane sequence of CD28,IgG1, CD4 or CD8a.

In some embodiments, the signaling domain sequence of the artificialCAR19 expression vector includes a repeated sequence of or anycombination sequence of the ITAM sequence or the CM sequence. That is,the numbers of the ITAM sequence or the CM sequence is at least two.

In one preferable embodiment, the transmembrane domain sequence of theartificial CAR19 expression vector is the transmembrane sequence ofCD8a.

In one preferable embodiment, the ITAM sequence of the signaling domainsequence of the artificial CAR19 expression vector is a CD3 zeta chain.

In some embodiments, the CM sequence of the artificial CAR19 expressionvector includes a CM sequence of CD27, CD28, CD30, 4-1BB/CD137, OX40 orHVEM. In another embodiment, the number of the CM sequence of thesignaling domain sequence is at least two, therefore the CM sequencesinclude a repeated sequence of or any combination sequence of the CMsequences of CD27, CD28, CD30, 4-1BB/CD137, OX40 or HVEM. In onepreferable embodiment, the artificial CAR19 expression vector includesone CM sequence, which is the CM sequence of 4-1BB/CD137 or CD28.

The present invention further relates to a pharmaceutical compositionincluding a population of genetically modified cell stably expresses atleast one artificial CAR19 protein or at least one expression vector ofthe artificial CAR19 with the specific properties as mentionedpreviously. In another embodiment, the genetically modified cell of thepharmaceutical composition not only stably express the at least oneartificial CAR19 protein but also the at least one expression vector ofthe artificial CAR19.

In another embodiment, the pharmaceutical composition consists of thepopulation of the genetically modified cell, the genetically modifiedcell stably expressed the at least one artificial CAR19 protein orincluded the at least one expression vector of the artificial CAR19. Inother words, the pharmaceutical composition only has the modified cellin it and without any other pharmaceutical component (e.g., cancerdrug). Furthermore, the modified cell may expresses the artificial CAR19protein, the artificial CAR19 expression vector or both.

In some embodiments, the modified cell used in the pharmaceuticalcomposition is a mammalian cell. In another embodiment, the modifiedcell used in the pharmaceutical composition is a lymphocyte.Furthermore, in one preferred embodiment, the modified cell used in thepharmaceutical composition is a CD3⁺ lymphocyte, for example, a T cell.

The present invention further relates to a therapeutic method oftreating a mammal having a disease, disorder or a condition associatedwith an elevated expression of an antigen. In one aspect, the diseasepreferably is cancer, e.g. CLL. In another aspect, the elevatedexpression of the antigen preferably is a CD19 antigen. The therapeuticmethod generally includes the following steps. In step (a), peripheralblood is isolated from at least one mammalian donor, and lymphocytes isfurther purified from the peripheral blood in step (b). In step (c), thepurified lymphocytes is further used to generate a pharmaceuticalcomposition. Step (d) relates to treating at least one mammalianrecipient with at least one chemotherapeutic agent. Then, administeringthe pharmaceutical composition to the at least one mammalian recipient.Specifically, the pharmaceutical composition in the therapeutic methodis the population of the lymphocytes that may express at least oneartificial CAR19 protein or carry at least one artificial CAR19expression vector with the specific properties as previously described.In some embodiments, the lymphocytes not only express the at least oneartificial CAR19 protein but also carries at least one artificial CAR19expression vector.

In another embodiment, the therapeutic method only consists the step (a)to the step (e) as the previously described. That is, there are onlyfive steps in the therapeutic method of treating a mammal having thedisease (e.g., cancer).

In some embodiments, the step (c) of the therapeutic method furtherincludes a step that amplifies the pharmaceutical composition.

In some embodiments, the pharmaceutical composition used in thetherapeutic method is autologous to at least one mammalian donor and onemammalian recipient. In other words, the modified lymphocytes of thepharmaceutical composition is purified from a mammalian donor and thenreadministrated back to the mammalian donor after engineering. Inanother embodiment, the pharmaceutical composition used in thetherapeutic method is allologous to at least one mammalian donor and onemammalian recipient. That is a mammalian donor is different from amammalian recipient.

In one preferred embodiment, the lymphocyte of the pharmaceuticalcomposition used in the therapeutic method is CD3⁺ lymphocyte (i.e., Tcells) or CD56⁺ lymphocyte (i.e., NK cells).

Definition

Throughout the various views and illustrative embodiments, likereference numerals are used to designate like elements. Reference willnow be made in detail to exemplary embodiments illustrated in theaccompanying drawings. Wherever possible, the same reference numbers areused in the drawings and the description to refer to the same or likeparts. In the drawings, the shape and thickness may be exaggerated forclarity and convenience. This description will be directed in particularto elements forming part of, or cooperating more directly with, anapparatus by the present disclosure. It is to be understood thatelements not specifically shown or described may take various forms.Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure, orcharacteristic described in connection with the embodiment is includedin at least one embodiment. Thus, the appearances of the phrases “Insome embodiments” or “in an embodiment” in various places throughoutthis specification are not necessarily all referring to the sameembodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments. It should be appreciated that the following figures are notdrawn to scale; rather, these figures are merely intended forillustration.

In the drawings, like reference numbers are used to designate like orsimilar elements throughout the various views, and illustrativeembodiments of the present disclosure are shown and described. Thefigures are not necessarily drawn to scale, and in some instances thedrawings have been exaggerated and/or simplified in places forillustrative purposes. One of ordinary skill in the art will appreciatethe many possible applications and variations of the present disclosurebased on the following illustrative embodiments of the presentdisclosure.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this disclosure belongs. It willbe further understood that terms; such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art and thepresent disclosure, and will not be interpreted in an idealized oroverly formal sense unless expressly so defined herein.

The term “about,” as used herein, when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

The term “activation,” as used herein, refers to the state of a T cellthat has been sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction, and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds to an antigen. Antibodies can beintact immunoglobulins derived from natural sources or recombinantsources and can be immunoreactive portions of intact immunoglobulins.Antibodies are typically tetramers of immunoglobulin molecules. Theantibodies of the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,Fv, Fab and F(ab)2, as well as single chain antibodies and humanizedantibodies.

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, F(ab′)2, and Fv fragments, linear antibodies, scFv antibodies,and multispecific antibodies formed from antibody fragments.

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations. κ and λ light chains refer tothe two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequence or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample. Such a biological sample can include, but isnot limited to a tissue sample, a tumor sample, a cell or a biologicalfluid.

The term “anti-tumor effect” or “anti-tumor” as used herein, refers to abiological effect which can be manifested by a decrease in tumor volume,a decrease in the number of tumor cells, a decrease in the number ofmetastases, an increase in life expectancy, or amelioration of variousphysiological symptoms associated with the cancerous condition. An“anti-tumor effect” can also be manifested by the ability of thepeptides, polynucleotides, cells and antibodies of the invention inprevention of the occurrence of tumor in the first place.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

As used herein, the term “allologous” is meant to refer to any materialderived from the different individual to which it is later to bere-introduced into the individual.

The term “allogeneic” refers to a graft derived from a different animalof the same species.

The term “xenogeneic” refers to a graft derived from an animal of adifferent species.

The term “cancer” as used herein is defined as a disease characterizedby the rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia, lung cancer and thelike.

“Co-stimulatory ligand,” as the term is used herein, includes a moleculeon an antigen presenting cell (e.g., dendritic cells, B cells, and thelike) that specifically binds a cognate co-stimulatory molecule on a Tcell and provides a signal. The signal, in addition to the primarysignal provided by binding of a TCR/CD3 complex with an MEW moleculeloaded with peptide, mediates a T cell response. The cell responseincludes, but not limited to, proliferation, activation,differentiation, and the like. A co-stimulatory ligand can include, butis not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL,OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist orantibody that binds Toll ligand receptor and a ligand that specificallybinds with B7-H3. A co-stimulatory ligand also encompasses, inter alia,an antibody that specifically binds with a co-stimulatory moleculepresent on a T cell, such as, but not limited to, CD27, CD28, 4-1BB,OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specificallybinds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds to a co-stimulatory ligand, therebymediating a co-stimulatory response by the T cell, such as, but notlimited to, proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class 1 molecule, BTLA and a Toll ligand receptor.

A “co-stimulatory signal”, as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or up-regulation or down regulation of keymolecules.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

An “effective amount” as used herein, means an amount which provides atherapeutic or prophylactic benefit.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or a mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes. The templates have either a defined sequence ofnucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of aminoacids and the biological properties resulting therefrom. Thus, a geneencodes a protein if transcription and translation of mRNA correspondingto that gene produces the protein in a cell or other biological system.Both the coding strand, the nucleotide sequence of which is identical tothe mRNA sequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

The term “immunoglobulin” or “Ig,” as used herein is defined as a classof proteins, which function as antibodies. Antibodies expressed by Bcells are sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present inbody secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingthe release of mediators from mast cells and basophils upon exposure tothe allergen.

“Isolated” as used herein means altered or removed from the naturalstate. For example, a nucleic acid or a peptide naturally present in aliving animal is not “isolated,” but the same nucleic acid or peptidepartially or completely separated from the coexisting materials of itsnatural state is “isolated.” An isolated nucleic acid or protein canexist in substantially purified form, or can exist in a non-nativeenvironment such as, for example, a host cell.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used, “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo.

The term “hyperexpression” of the tumor antigen is intended to indicatean abnormal level of expression of the tumor antigen in a cell from adisease area like a solid tumor within a specific tissue or organ of thepatient relative to the level of expression in a normal cell from thattissue or organ. Patients having solid tumors or a hematologicalmalignancy characterized by overexpression of the tumor antigen can bedetermined by standard assays known in the art.

The term “administration of” or “administering” an immunogeniccomposition includes, e.g., subcutaneous (s.c.), intravenous (i.v.),intramuscular (i.m.), or intrasternal injection, or infusion techniques.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In certain non-limiting embodiments, the patient, subject or individualis a human.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types, “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

By the term “specifically binds,” as used herein concerning an antibody,is meant an antibody which recognizes a specific antigen, but does notsubstantially recognize or bind other molecules in a sample. Forexample, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. However,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

The term “stimulation” or “amplification” is meant a primary responseinduced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex)with its cognate ligand thereby mediating a signal transduction event,such as, but not limited to, signal transduction via the TCR/CD3complex. Moreover, the term “stimulate” or “amplify” is used to describethe process that using the stimulatory ligand to trigger the primaryresponse. Further, the terms “stimulate” and “amplify” areinterchangeable. Stimulation can mediate altered expressions of certainmolecules, such as down-regulation of TGF-β, and/or reorganization ofcytoskeletal structures, and the like.

The term “generating” or “amplifying” a composition, means to increasethe quantity of the composition, e.g. a cell number. Hence, the term“generating” and “amplifying” are interchangeable.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds to a cognate stimulatory ligandpresent on an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an APC, a dendritic cell, a B-cell,and the like) can specifically bind with a cognate binding partner(referred to herein as a “stimulatory molecule”) on a T cell, therebymediating a primary response by the T cell, including, but not limitedto, activation, initiation of an immune response, proliferation, and thelike. Stimulatory ligands are well-known in the art and encompass, intercilia, an MHC Class I molecule loaded with a peptide, an anti-CD3antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2antibody.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals). Examples of subjectsinclude humans, dogs, cats, mice, rats, and transgenic species thereof.

As used herein, a “purified” cell is a cell that is essentially free ofother cell types. A substantially purified cell also refers to a cellwhich has been separated from other cell types with which it is normallyassociated in its naturally occurring state. In some instances, apopulation of substantially purified cells refers to a homogenouspopulation of cells. In other instances, this term refers simply to acell that has been separated from the cells with which they arenaturally associated in their natural state. In some embodiments, thecells are cultured in vitro. In other embodiments, the cells are notcultured in vitro.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. Therefore, as describing the transferprocess, these three words are interchangeable. A “transfected” or“transformed” or “transduced” cell is one which has been transfected,transformed or transduced with exogenous nucleic acid. The cell includesthe primary subject cell and its progeny.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate the transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

Description

The present invention provides compositions and a method for treating adisease, disorder or a condition. More specifically, the diseaseincludes cancer which may be a hematological malignancy, a solid tumor,a primary or a metastasis tumor. Preferably, the cancer is ahematological malignancy, and more preferably, the cancer is ChronicLymphocytic Leukemia (CLL). Other diseases that may be treated with theadministration of the compositions of the present invention includeviral, bacterial, parasitic infections or autoimmune diseases.

Protein Composition

One of the compositions of the present invention is an artificial CARprotein that includes three portions, i.e. an extracellular domain, atransmembrane domain and an intracellular domain. The extracellulardomain includes a target-specific binding element otherwise referred toas an antigen binding moiety. The transmembrane domain links theextracellular domain and the intracellular domain, while also supportsthe extracellular domain on the cell surface. The intracellular domainor otherwise the cytoplasmic domain includes a signaling domain. Thesignaling domain further includes at least one ITAM region, at least oneCM, or any combination thereof. The CM is a cell molecule other than anantigen receptor that is required for an efficient response oflymphocytes to antigen.

Within the extracellular domain or the cytoplasmic domain of the CAR,between the extracellular domain and the transmembrane domain of theCAR, or between the cytoplasmic domain and the transmembrane domain ofthe CAR, at least one linker or spacer domain may be incorporated. Asused herein, the term “linker” generally means any oligo-or polypeptidethat functions to link protein domains in the polypeptide chain. Alinker may include about 300 amino acids, preferably about 10 to 100amino acids, preferably about 10 to 50 amino acids and most preferablyabout 25 to 50 amino acids.

Antigen Binding Moiety

In some embodiments, the CAR of the invention comprises atarget-specific binding element otherwise referred to as an antigenbinding moiety that can be recognized by a synthetic antibody. Thechoice of moiety depends upon the type and number of ligands that definethe surface of a target cell. For example, the antigen binding domainmay recognize a ligand that acts as a cell surface marker on targetcells associated with a particular disease state. Examples of cellsurface markers that may act as ligands for the antigen binding moietyin the CAR of the present invention include those associated with viral,bacterial and parasitic infections, autoimmune disease and cancer cells.

In some embodiments, the CAR of the present invention can be engineeredto target a tumor antigen of interest by way of engineering a desiredantigen binding moiety that specifically binds to an antigen on a tumorcell. In the context of the present invention, “tumor antigen” or“hyper-expression disorder antigen” or “antigen associated with ahyper-expression disorder,” refers to antigens that are common tospecific hyper-expression disorders such as cancer. The antigensdiscussed herein are merely included as examples. The list is notintended to be exclusive and other examples are readily apparent tothose of skill in the art.

Tumor antigens are proteins that are produced by tumor cells that elicitan immune response, particularly T-cell mediated immune response. Theselection of the antigen binding moiety of the present invention willdepend on the particular type of cancer to be treated. In someembodiments, the tumor antigen comprises one or more antigenic cancerepitopes associated with a malignant tumor. Malignant tumors expressnumbers of proteins that can serve as target antigens for an immuneattack. These molecules include but are not limited to tissue-specificantigens such as MART-1, tyrosinase and GP 100 in melanoma and prostaticacid phosphatase (PAP) and prostate-specific antigen (PSA) in prostatecancer. Other target molecules belong to the group oftransformation-related molecules such as oncogene HER-2/Neu/ErbB-2.Another group of target antigens is onco-fetal antigens such ascarcinoembryonic antigen (CEA). In B-cell lymphoma, the tumor-specificidiotype immunoglobulin constitutes a truly tumor-specificimmunoglobulin antigen that is unique to the individual tumor. B-celldifferentiation antigens such as hemagglutinin protein of influenzavirus, hen egg lysozyme, ovalbumin, CD19, CD20 and CD37 are othercandidates for target antigens in B-cell lymphoma. Some of theseantigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targetsfor passive immunotherapy with monoclonal antibodies with limitedsuccess.

The type of tumor antigen referred to in the present invention may alsobe a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). ATSA is unique to tumor cells and does not express on other cells in thebody. A TAA associated antigen is not unique to a tumor cell and insteadis also expressed on a normal cell under conditions that fail to inducea state of immunologic tolerance to the antigen. The expression of theantigen on the tumor may occur under conditions that enable the immunesystem to respond to the antigen. TAAs may be antigens that areexpressed on normal cells during fetal development when the immunesystem is immature and unable to respond or they may be antigens thatpresent at extremely low levels in normal cells but which are expressedat much higher levels in tumor cells.

Depending on the desired antigen to be targeted, the CAR of the presentinvention can be engineered to include the appropriate antigen bindmoiety that is specific to the desired antigen target. For example, ifCD19 is the desired antigen, which is the target antigen, an antibodyfor CD19 can be used as the antigen bind moiety to be incorporated intothe CAR of the present invention.

Transmembrane Domain

The CAR of the present invention can be designed to comprise atransmembrane domain that is fused to the extracellular domain (e.g.,the antigen binding moiety) of the CAR. In some embodiments, thetransmembrane domain that naturally associates with one of the domainsin the CAR is used. For example, the transmembrane domain can beselected or modified by amino acid substitution to avoid binding of suchdomains to the transmembrane domains of the same or different surfacemembrane proteins to minimize interactions with other members of thereceptor complex. In another embodiment, the transmembrane domain doesnot directly connect or link to any domain of the antigen bind moiety.Therefore, there is at least one short peptide (e.g., linker) betweenthe transmembrane domain and the antigen bind moiety.

The transmembrane domain may be derived either from a natural or asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Transmembrane regionsof particular use in this invention may be derived from (i.e. compriseat least the transmembrane region(s) of) the alpha, beta or zeta chainof the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, IgG1,CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.Alternatively the transmembrane domain may be synthetic, in which caseit will comprise predominantly hydrophobic residues such as leucine andvaline. Preferably a triplet of phenylalanine, tryptophan and valinewill be found at each end of a synthetic transmembrane domain.Optionally, a short oligo- or polypeptide linker, preferably between 2and 10 amino acids in length may form the linkage between thetransmembrane domain and the cytoplasmic signaling domain of the CAR. Aglycine-serine doublet provides a particularly suitable linker.

In some embodiments, the transmembrane domain in the CAR of the presentinvention is CD28, IgG1, CD4, CD8α or any combination thereof. In apreferred embodiment, the transmembrane domain of the CAR is thetransmembrane domain of CD8a.

Signaling Domain

The signaling domain or otherwise the cytoplasmic domain of the CAR ofthe present invention is responsible for activating at least one of thenormal effector functions of the immune cell. The term “effectorfunction” refers to a specialized immune function of an immune cell.Effector function of a lymphocyte cell, more specifically a CD3⁺lymphocyte, may be a cytolytic activity or helper activity including thesecretion of cytokines. The term “signaling domain” refers to theportion of a protein which transduces the effector function signal anddirects the cell to perform a specialized function. While usually theentire intracellular signaling domain can be employed, in many cases itis not necessary to use the entire protein. To the area that is beingused as a truncated portion of the signaling domain, such truncatedportion may be used in place of the whole protein as long as ittransduces the effector function signal. Therefore, the term signalingdomain is meant to include any truncated portion of the signaling domainsufficient to transduce the effector function signal.

Preferred examples of signaling domains for use in the CAR of thepresent invention include the cytoplasmic sequences of the T cellreceptor (TCR) and co-receptors. Both of these domain act in concert toinitiate signal transduction following antigen receptor engagement, aswell as any derivative or variant of these sequences and any syntheticsequence that has the same functional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation ismediated by two distinct classes of signaling sequence: those thatinitiate antigen-dependent primary activation through the TCR (primarysignaling sequences) and those that act in an antigen-independent mannerto provide a secondary or co-stimulatory signal (secondary signalingsequences).

Primary signaling domains regulate primary activation of the TCR complexeither in a stimulatory way, or in an inhibitory way. Primary signalingdomains that act in a stimulatory manner may contain signaling motifswhich are known as ITAMs. The secondary or co-stimulatory signalingdomains that act in a stimulatory manner may contain signaling motifswhich are known as CMs.

Examples of ITAM containing primary signaling domains that are ofparticular use in the present invention include those derived from TCRzeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22,CD79a, CD79b, and CD66d. Preferably, the signaling molecule (i.e. ITAM)of the CAR of the present invention comprises a signaling sequencederived from CD3 zeta.

In a preferred embodiment, the ITAM of the signaling domain of the CARcan be designed to comprise the CD3-zeta signaling domain by itself orcombined with any other desired cytoplasmic domain(s) useful in thecontext of the CAR of the present invention. For example, thecytoplasmic domain of the CAR can comprise a CD3 zeta chain portion anda costimulatory signaling region. The costimulatory signaling regionrefers to a portion of the CAR comprising the intracellular domain ofthe CM. The CM is a cell molecule other than an antigen receptor thatare required for an efficient response of lymphocytes to an antigen.Examples of such molecules include CD27, CD28, 4-1BB/CD137, OX40, CD30,CD40, PD-1, ICOS, HVEM, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specificallybinds to CD83, and the like. Thus, while the present invention involvesprimarily with 4-1BB/CD137 as the co-stimulatory signaling molecule,other costimulatory molecules are within the scope of the invention.

The ITAM and the CM within the cytoplasmic signaling domain portion ofthe CAR of the invention may be linked to each other in a random orspecified order. Optionally, a short oligo- or polypeptide linker,preferably about between 2 and 10 amino acids in length may form thelinkage. A glycine-serine doublet provides a particularly suitablelinker.

In some embodiments, the signaling domain is designed to comprise notonly the ITAM but also the CM domain including CD27, CD28, CD30,4-1BB/CD137, OX40, HVEM or any combination thereof. In anotherembodiment, the CM of the signaling domain in the intracellular portionof the CAR comprises 4-1BB/CD137. In a preferred embodiment, thesignaling domain is designed to comprise the ITAM of CD3-zeta and the CMof 4-1BB/CD137.

Vectors

The present invention encompasses a DNA vector construction comprising anucleic acid sequence of CAR. Further, the nucleic acid sequenceincludes the nucleic acid sequence of an antigen-binding fragmentoperably linked to the nucleic acid sequence of a transmembrane domainand a signaling domain. The nucleic acid sequences coding for thedesired molecules can be obtained using recombinant methods known in theart, for example by screening libraries from cells expressing the gene,by deriving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically, rather than cloned.

The present invention also provides vectors for inserting a DNA of thepresent invention. The vectors derived from retroviruses such as thelentivirus are suitable tools to achieve long-term gene transfer sincethey allow long-term, stable integration of a transgene and itspropagation in daughter cells. Lentiviral vectors have the addedadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-dividing cells, such ashepatocytes. They also have the added advantage of low immunogenicity.

Briefly, the expression of natural or synthetic nucleic acids encodingCARs is achieved by operably linking a nucleic acid encoding the CARpolypeptide or portions thereof to a promoter, and incorporating theconstruct into an expression vector. The vectors can be suitable forreplication and integration eukaryotes. Typical cloning vectors containtranscription and translation terminators, initiation sequences, andpromoters useful for regulation of the expression of the desired nucleicacid sequence.

The expression constructs of the present invention may also be used fornucleic acid immunization and gene therapy, under standard gene deliveryprotocols. In another embodiment, the invention provides a gene therapyvector.

The nucleic acid can be cloned into some types of vectors. For example,the nucleic acid can be cloned into a vector including, but not limitedto a plasmid, a phagemid, a phage derivative, an animal virus, and acosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be infected into a cell in the formof a viral vector. Viral vector technology is well known in the art.Viruses, which are useful as vectors include, but are not limited to,retroviruses, adenoviruses, adeno-associated viruses, herpes viruses,and lentiviruses. Generally, a suitable vector contains an origin ofreplication functional in at least one organism, a promoter sequence,convenient restriction endonuclease sites, and one or more selectable.

Some viral based systems have been developed for gene transfer intomammalian cells. For example, retroviruses provide a convenient platformfor gene delivery systems. A selected gene can be inserted into a vectorand packaged in retroviral particles using techniques known in the art.The recombinant virus can then be isolated and delivered to cells of thesubject either in vivo or ex vivo. Some retroviral systems are known inthe art. In some embodiments, lentivirus vectors are used.

In order to assess the expression of CAR, the expression vector to beintroduced into a cell can also contain either a selectable marker geneor a reporter gene or both to facilitate identification and selection ofexpressing cells from the population of cells sought to be transfectedor infected through viral vectors. In other aspects, the selectablemarker may be carried on a separate piece of DNA and used in aco-transfection procedure. Both selectable markers and reporter genesmay be flanked with appropriate regulatory sequences to enableexpression in the host cells. Useful selectable markers include, forexample, antibiotic-resistance genes, such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein (GFP) gene.Suitable expression systems are well known and may be prepared usingknown techniques or obtained commercially. In general, the constructwith the minimal 5′ flanking region showing the highest level ofexpression of reporter gene is identified as the promoter. Such promoterregions may be linked to a reporter gene and used to evaluate agents forthe ability to modulate promoter-driven transcription. In someembodiments, the GFP is used.

Methods of introducing and expressing genes in a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, gene gun, and the like.Methods for producing cells comprising vectors and/or exogenous nucleicacids are well-known in the art. A preferred method for the introductionof a polynucleotide into a host cell is electroporation transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from a lentivirus, poxviruses, herpes simplexvirus I, adenoviruses and adeno-associated viruses, and the like.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

Regardless of the method used to introduce exogenous nucleic acids intoa host cell, to confirm the presence of the recombinant DNA sequence inthe host cell, a variety of assays may be performed. Such assaysinclude, for example, “molecular biological” assays well known to thoseof skill in the art, such as Southern and Northern blotting, RT-PCR andPCR; “biochemical” assays, such as detecting the presence or absence ofa particular peptide, e.g., by immunological means (ELISAs and Westernblots) or by assays described herein to identify agents falling withinthe scope of the invention.

Pharmaceutical Composition

The invention further provides a pharmaceutical composition including apopulation of a cell (e.g., lymphocyte) expressed at least one ofartificial CAR protein or contained at least one of DNA vectorconstruction of the artificial CAR. Therefore, the CAR cell exhibits anantitumor property.

Sources of Cells

Before expansion and genetic modification of the CD3⁺ lymphocytes of theinvention, a source of CD3⁺ Lymphocytes is obtained from a subject. CD3⁺Lymphocytes can be obtained from numbers of sources, includingperipheral blood mononuclear cells, bone marrow, lymph node tissue, cordblood, thymus tissue, tissue from a site of infection, ascites, pleuraleffusion, spleen tissue, and tumors. In certain embodiments of thepresent invention, any number of CD3⁺ Lymphocytes available in the art,may be used. In certain embodiments of the present invention, CD3⁺Lymphocytes can be obtained from a unit of blood collected from asubject using any number of techniques known to the skilled artisan,such as Ficoll™ separation. In one preferred embodiment, cells from thecirculating blood of an individual are obtained by apheresis. Theapheresis product typically contains lymphocytes, including T cells,monocytes, granulocytes, B cells, other nucleated white blood cells, redblood cells, and platelets. In some embodiments, the cells collected byapheresis may be washed to remove the plasma fraction and to place thecells in an appropriate buffer or media for subsequent processing steps.In some embodiments of the invention, the cells are washed withphosphate buffered saline (PBS). After washing, the cells may bere-suspended in a variety of biocompatible buffers, such as, forexample, Ca²⁺-free, Mg²⁺-free PBS, or other saline solution with orwithout buffer. Alternatively, the undesirable components of theapheresis sample may be removed and the cells directly re-suspended inculture media.

In another embodiment, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL™ gradient. A specificsubpopulation of T cells, such as CD2⁺ and CD3⁺, can be further isolatedby positive or negative selection techniques. For example, In someembodiments, T cells are isolated by incubation with anti-CD14,anti-CD16, anti-CD19, anti-CD36, anti-CD56, anti-CD123 andanti-Glycophorin A conjugated beads, such as MACS® Pan T Cell IsolationKit II, for a time period sufficient for negative selection of thedesired T cells. In certain embodiments, it may be desirable to performthe selection procedure and use the “unselected” cells in the activationand expansion process. “Unselected” cells can also be subjected tofurther rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic or flow cytometry thatuses a cocktail of monoclonal antibodies directed to cell surfacemarkers present on the cells negatively selected.

The source of the cells to be expanded can be collected at any timepoint necessary, and desired cells, such as T cells, isolated and frozenfor later use in T cell therapy for any number of diseases or conditionsthat would benefit from T cell therapy, such as those described herein.In some embodiments, a blood sample or an apheresis is taken from ahealthy subject. In another embodiment, a blood sample or an apheresisis taken from a healthy subject who is at risk of developing a disease,but who have not yet developed a disease, and the cells of interest areisolated and frozen for later use. In certain embodiments, the T cellsmay be expanded, frozen, and used at a later time. In certainembodiments, samples are collected from a patient shortly afterdiagnosis of a particular disease as described herein but before anytreatments. In a further embodiment, the cells are isolated from a bloodsample or an apheresis from a subject prior to any number of relevanttreatment modalities, including but not limited to treatment with agentssuch as natalizumab, efalizumab, antiviral agents, chemotherapy,radiation, immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan,fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids,FR901228, and irradiation. In a further embodiment, the cells areisolated from a patient and frozen for later use in conjunction with(e.g., before, simultaneously or following) bone marrow or stem celltransplantation, T cell ablative therapy using either chemotherapyagents such as, fludarabine, external-beam radiation therapy (XRT),cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In anotherembodiment, the cells are isolated prior to and can be frozen for lateruse for treatment following B-cell ablative therapy such as agents thatreact with CD20, e.g., Rituxan.

Activation and Expansion of T Cells

Whether before or after genetic modification of the T cells to express adesirable CAR, the T cells can be activated and expanded using methodsas described.

The T cells of the invention are expanded by contact with a surfacehaving attached to it an agent that stimulates a CD3/TCR complexassociated signal and a ligand that stimulates a co-stimulatory moleculeon the surface of the T cells. In particular, T cell populations may bestimulated as described herein, such as by contact with an anti-CD3antibody, or antigen-binding fragment thereof, or an anti-CD2 antibodyimmobilized on a surface, or by contact with a protein kinase Cactivator (e.g., bryostatin) in conjunction with a calcium ionophore.For co-stimulation of an accessory molecule on the surface of the Tcells, a ligand that binds the accessory molecule is used. For example,a population of T cells can be contacted with an anti-CD3 antibody andan anti-CD28 antibody, under conditions appropriate for stimulatingproliferation of the T cells. To stimulate proliferation of either CD4⁺or CD8⁺ T cells, an anti-CD3 antibody and an anti-CD28 antibody areused. Examples of an anti-CD3 antibody include HIT3a, UCHT1 and OKT3 (BDPharmingen™ USA) can be used as can other methods commonly known in theart (Kemper et al., Nature 421.6921: 388-92. 2003; Li et al., J.Immunother. 35(2):189-95, 2012). Examples of an anti-CD28 antibodyinclude CD28.2, L293 and clone 15E8 (BD Pharmingen™, USA) can be used ascan other methods commonly known in the art as previous mention.

In some embodiments, the two agents are immobilized on beads, either onthe same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By wayof example, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the co-stimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts.

In further embodiments of the present invention, the cells, such as Tcells, are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the agent-coated beads andcells are not separated but are cultured together. In a furtherembodiment, the beads and cells are first concentrated by application offorce, such as a magnetic force, resulting in increased ligation of cellsurface markers, thereby inducing cell stimulation.

In some embodiments of the present invention, the mixture may becultured for several hours (about 3 hours) to about 14 days or anyhourly integer value in between. In another embodiment, the mixture maybe cultured for 21 days. In some embodiments of the invention the beadsand the T cells are cultured together for about eight days. In anotherembodiment, the beads and T cells are cultured together for 2-3 days.Several cycles of stimulation may also be desired such that culture timeof T cells can be 60 days or more. Conditions appropriate for T cellculture include an appropriate media (e.g., RPMI Media 1640 or LGM-3™Lymphocyte Growth Medium (Lonza) or AIM-V) that may contain factorsnecessary for proliferation and viability, including serum (e.g., fetalbovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4,IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α or any otheradditives for the growth of cells known to the skilled in the art. Otheradditives for the growth of cells include, but are not limited to,surfactant, plasmanate, and reducing agents such as N-acetyl-cysteineand 2-mercaptoethanol. Media can include RPMI 1640, DMEM, MEM, α-MEM,and F-12, Optimizer, with added amino acids, sodium pyruvate, andvitamins, either serum-free or supplemented with an appropriate amountof serum (or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO2).

T cells exposed to varied stimulation times may exhibit differentcharacteristics. For example, typical blood or apheresed peripheralblood mononuclear cell products have a helper T cell population (TH,CD4⁺) that is greater than the cytotoxic or suppressor T cell population(Tc, CD8⁺). Ex vivo expansion of T cells by stimulating CD3 and CD28receptors produces a population of T cells that prior to about days 8-9consists predominately of TH cells, while after about days 8-9, thepopulation of T cells comprises an increasingly greater population of Tccells. Accordingly, depending on the purpose of treatment, infusing asubject with a T cell population comprising predominately of TH cellsmay be advantageous. Similarly, if an antigen-specific subset of Tccells has been isolated it may be beneficial to expand this subset to agreater degree.

Therapeutic Application

The present invention encompasses a cell (e.g., T cell) transduced witha lentiviral vector (LV). For example, the LV encodes a CAR thatcombines an antigen recognition domain of a single-chain variablefragment, a transmembrane domain of CD8α and a signaling domain of CD3zeta, 4-1BB/CD137, CD28 or any combinations thereof. Therefore, in someinstances, the transduced T cell can elicit a CAR-mediated T-cellresponse.

The invention provides the use of a CAR to redirect the specificity of aprimary T cell to a tumor antigen. Thus, the present invention alsoprovides a method for stimulating a T cell-mediated immune response to atarget cell population or tissue in a mammal comprising the step ofadministering to the mammal with a popution of a T cell expressed a CAR.

In some embodiments, the present invention includes a type of cellulartherapy where T cells are genetically modified to express a CAR and theCAR T cell is infused to a recipient in need thereof. The infused cellcan kill tumor cells in the recipient. Unlike antibody therapies, CAR Tcells can replicate in vivo resulting in long-term persistence that canlead to sustained tumor control.

In some embodiments, the CAR T cells of the invention can undergo robustin vivo T cell expansion and can persist for an extended amount of time.In another embodiment, the CAR T cells of the invention evolve intospecific memory T cells that can be reactivated to inhibit anyadditional tumor formation or growth. The anti-tumor immunity responseelicited by the CAR-modified T cells may be an active or a passiveimmune response. Also, the CAR mediated immune response may be part ofan adoptive immunotherapy approach in which CAR-modified T cells inducean immune response specific to the antigen binding moiety in the CAR.For example, a CAR19 T cells elicits an immune response specific againstcells expressing CD19.

While the data disclosed herein specifically disclose lentiviral vectorcomprising anti-CD19 scFv derived from a monoclonal antibody, human CD8αhinge and transmembrane domain, and human 4-1BB/CD137 and CD3 zetasignaling domains, the invention should be construed to include anynumber of variations for each of the components of the construct asdescribed elsewhere herein. That is, the invention includes the use ofany antigen binding moiety in the CAR to generate a CAR-mediated T-cellresponse specific to the antigen binding moiety. For example, theantigen binding moiety in the CAR of the invention can target a tumorantigen for treating cancer.

Cancers that may be treated include tumors that are not vascularized, ornot yet substantially vascularized, as well as vascularized tumors. Thecancers may comprise non-solid tumors (such as hematological tumors, forexample, leukemias and lymphomas) or may comprise solid tumors.

In some embodiments, the antigen bind moiety portion of the CAR of theinvention is designed to treat a particular cancer. For example, the CARdesigned to target CD19 can be used to treat cancers and disordersincluding but are not limited to pre-B ALL (pediatric indication), adultALL, mantle cell lymphoma, diffuse large B-cell lymphoma, salvage postallogenic bone marrow transplantation, and the like. In anotherembodiment, the cancers and the disorders can be treated using acombination of CARs that target CD19, CD20, CD22, and ROR1.

The CAR modified T cells of the invention may also serve as a type ofvaccine for ex vivo immunization and/or in vivo therapy in a mammal.Preferably, the mammal is a human.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (preferably ahuman) and genetically modified (i.e., transduced or transfected invitro) with a vector expressing a CAR disclosed herein. The CAR-modifiedcell can be administered to a mammalian recipient to provide atherapeutic benefit. The mammalian recipient may be a human and themodified CAR cell can be autologous with respect to the recipient.Alternatively, the cells can be allogeneic, syngeneic or xenogeneic withrespect to the recipient.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, the CAR modified Tcells of the invention are used in the treatment of CLL. In certainembodiments, the cells of the invention are used in the treatment ofpatients at risk for developing CLL. Thus, the present inventionprovides methods for the treatment or prevention of CLL comprisingadministering to a subject in need thereof, a therapeutically effectiveamount of the CAR-modified T cells of the invention.

The CAR modified T cells of the present invention may be administeredeither alone, or as a pharmaceutical composition in combination withdiluents and/or with other components such as IL-2 or other cytokines orcell populations. Briefly, pharmaceutical compositions of the presentinvention may comprise a target cell population as described herein, incombination with one or more pharmaceutically or physiologicallyacceptable carriers, diluents or excipients. Such compositions maycomprise buffers such as neutral buffered saline, phosphate bufferedsaline and the like.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials. The optimal dosage and treatment regime for aparticular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

In certain embodiments, it may be desired to administer activated Tcells to a subject and then subsequently redraw blood (or have anapheresis performed), activate T cells therefrom according to thepresent invention, and reinfuse the patient with these activated andexpanded T cells. This process can be carried out multiple times everyfew weeks.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patientsubcutaneously, intradermally, intratumorally, intramuscularly, byintravenous (i.v.) injection, or intraperitoneally (i.p.). In someembodiments, the T cell compositions of the present invention areadministered to a patient by intradermal or subcutaneous injection. Inanother embodiment, the compositions of T cells may be injected directlyinto a tumor, lymph node, or site of infection.

In further embodiments, the T cells may be used in combination withchemotherapy, radiation, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAMPATH, anti-CD3 antibodies orother antibody therapies, cytoxin, fludaribine, cyclosporin, rapamycin,mycophenolic acid, steroids, FR901228, cytokines, and irradiation. In afurther embodiment, the cell compositions of the present invention areadministered to a patient in conjunction with (e.g., before,simultaneously or following) bone marrow transplantation, T cellablative therapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH. In another embodiment, the cell compositions ofthe present invention are administered following B-cell ablative therapysuch as agents that react with CD20, e.g., Rituxan. In another certainembodiment, the used chemotherapy protocol can select from the followingprocedures: (A) Flu/Cy-A: administrating Fludarabine 30 mg/m² daily for3 days and Cyclophosphamide 300 mg/m² daily for 3 days; (B) Flu/Cy-B:administrating Fludarabine 30 mg/m² daily for 4 days andCyclophosphamide 500 mg/m² daily for 2 days; or (C) Flu/Cy-C:administrating Fludarabine 25 mg/m² daily for 3 days andCyclophosphamide 60 mg/m² daily for 1 days.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices.

Experimental Example

The invention is further described in detail by reference to thefollowing experimental example. This example is provided for purposes ofillustration only, and is not intended to be limiting unless otherwisespecified. Thus, the invention should in no way be construed as beinglimited to the following example, but rather, should be construed toencompass any and all variations which become evident as a result of theteaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexample therefore, specifically points out the preferred embodiments ofthe present invention, and is not to be construed as limiting in any waythe remainder of the disclosure.

The materials and methods employed in these experiments are nowdescribed.

Material and Method

Anti-CD19 CAR Lentiviral Vector Production

The clones of the mouse anti-human CD19 scFvs were generated by UwellBiopharma. Codon optimization of the anti-CD19 scFv sequences wasperformed to improve gene expression in human cells. According to thegene sequence of NCBI GenBank, these plasmids,anti-CD19-scFv-CD8α-CD137-CD3ζ were designed. All the sequences of theplasmids were confirmed by DNA sequencing (Genomics, Taiwan).

Lentiviral Production and Determination of Viral Titer

The HEK 293T cells were transfected with the lentiviral transfer vectorDNA, together with the packaging plasmids pCMVdeltaR8.91 and pMD.G(Academia Sinica, Taiwan) using X-tremeGENE 9 (Roche, Basel,Switzerland). The supernatant containing lentiviral particles wascollected and concentrated using Lenti-X Concentrator (ClontechLaboratories, Mountain View, Calif.). The viruses were aliquoted andfrozen at −80° C. until ready to use for virus tittering. All thelentiviruses used in the experiments were from concentrated frozenstocks. Titers of the lentivirus were determined by SUP-T1 cells basedon 3-fold serial dilution of the concentrated lentivirus. Shortly, 50 ul3-fold serial diluted concentrated lentivirus from 1:3 to a finaldilution of 1:2,187 ul per well was transferred to SUP-T1 cells (20,000cells/100 ul/well) seeded in a single well of a 96-well plate andincubated overnight. Two days post transduction, the SUP-T1 cells werestained with Biotin-SP-conjugated AffiniPure Goat anti-mouse IgG,F(ab′)2fragment specific (Jackson ImmunoResearch, West Grove, Pa.) followed byPE-conjugated streptavidin (Jackson ImmunoResearch, West Grove, Pa.).The percentage of CAR-positive cells was analyzed using flow cytometry(FACSCalibur cytometer, Becton Dickinson) and FlowJo software. Onlydilutions generating 1 to 20% of CAR-positive cells should be consideredfor titer calculation. The percentage of CAR-positive cells ismultiplied by the number of cells (20,000) seeded per well, then dividedby the actual volume of added crude supernatant (in ml) to determine thetiter of lentivirus in the supernatant (transducing units/ml).Multiplicity of infection (MOI) is defined as the ratio of transducingunits of viral particles to the number of target T cells.

Cell Lines

The HEK 293T cells and human leukemia cell lines SUP-T1, Raji and RS4;11cells were purchased from the American Type Culture Collection (ATCC;Rockville, Md.). The SUP-T1 cells were used for lentivirus titeranalysis. The human leukemia cell line K562 was purchased from theBioresource Collection and Research Center (BCRC; Hsinchu, Taiwan). TheRaji, RS4;11, SUP-T1 and K562 cells were maintained in RPMI-1640(Invitrogen, Carlsbad, Calif.) and the HEK 293T cells was maintained inDMEM (HyClone; GE Healthcare, South Logan, Utah); media weresupplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, Calif.)and 100 mg/mL penicillin/streptomycin (Gibco, Carlsbad, Calif.).

Preparation of Fluorescence Tumor Cells

For the visualization of injected tumor cells in immunodeficient mice,the Raji cells were transduced with aLentivirus-EF1alpha-MCS-IRES-Luciferase plasmid (Addgene, Cambridge,Mass.) and the single Raji-Luc cell with the highest expression ofluciferase was sorted with a FACSAria cell sorter (BD Biosciences, SanJose, Calif.).

Human T Cells Transduction

Peripheral blood samples were obtained from discarded anonymizedby-products of platelet donations from healthy adult donors granted bythe Institutional Review Board of Kaohsiung Veterans General Hospital(Protocol number: VGHKS13-CT6-11). Mononuclear cells collected from thesamples by centrifugation on a Lymphoprep density step (Nycomed, Oslo,Norway) were washed twice using medium. Human T cells were purified bynegative selection with a mixture of CD14, CD16, CD19, CD36, CD56,CD123, and CD235a antibodies and magnetic separation (Pan T CellIsolation Kit II; Miltenyi Biotec). The percentage of CD3 and CD56expression of T cells was determined with CD3-PerCP and CD56-FITC (BDBiosciences, San Jose, Calif.) staining using flow cytometry(FACSCalibur cytometer, Becton Dickinson). Purified T cells weremaintained in RPMI-1640 medium containing 10% FBS and recombinant humanIL-2 (rHuIL-2, 50 IU/ml; Proleukin, Novartis Pharma) with anti-CD3/CD28beads (Thermo Fisher Scientific, Carlsbad, Calif.) until virustransduction. Within 24 hours, T cells were transduced with thawedlentiviruses using the same MOI to generate the CAR-T cells possessingthe different scFv-derived binding domains. The culture medium waschanged every 2 days and then the cells were transferred to G-Rex24-well culture plate (Wolson Wolf Manufacturing, Saint Paul, Minn.) fora total 8-day expansion culture. Further, the cells were transferred toG-Rex 24-well culture plate after culturing four days (i.e., at thefifth day) and harvest at the eighth day. The T cells were stained withBiotin-SP-conjugated AffiniPure Goat anti-mouse IgG,F(ab′)2 fragmentspecific (Jackson ImmunoResearch, West Grove, Pa.) followed byPE-conjugated streptavidin (Jackson ImmunoResearch, West Grove, Pa.) todetermine the percentage of CAR-positive T cells.

In Vitro Functional Assays

To verify the specific antitumor capacity of anti-CD19 CAR-T cellsagainst B-cell malignancy, CD19 positive RS4;11 and Raji cells were usedas target cells, whereas CD19 negative K562 cells was regarded ascontrol cells. The anti-CD19 CAR-T cells and target cells wereco-cultured in 96-well plates with indicated E:T ratios. After 24 hours,cells were harvested, washed and stained with PE-conjugated mouseanti-human CD19 antibody (BD Biosciences, San Jose, Calif.). Thepercentage of CD19 positive cells representing the level of residualleukemia cells was analyzed by FACSCalibur flow cytometry.

The supernatants were harvested for the analysis of IFN-γ production ofthe CAR-T cells in response to leukemia cells using ELISA kits (R&DSystems, Inc, Minneapolis, Minn.). The absorbance at 450 nm in each wellwas measured using a microplate reader (Anthos 2010, Biochrom Ltd.,Cambridge, UK). Cytotoxicity assay was further analyzed to determine thespecific anti-leukemia ability of CAR-T cells. The target cells weresuspended in RPMI-1640 containing 10% FBS, labeled with calcein AM (BDBiosciences, San Jose, Calif.), and plated onto 96-well flat-bottomplates (Costar, Corning, N.Y.). The CAR-T cells, suspended in RPMI-1640containing 10% FBS, were then added at various E:T ratios andco-cultured with the target cells for 4 h. Following this, the cellswere stained with propidium iodide (Sigma-Aldrich, St. Louis, Mo.), andthe cytotoxicity was assessed by flow cytometry on a FACSCalibur (BectonDickinson) instrument enumerating the number of viable target cells(calcein AM-positive, propidium-iodide negative, and light scatteringproperties of viable cells).

Multi-Parameter Flow Cytometry

Cells were evaluated by flow cytometry either fresh after Ficoll-Paqueprocessing or frozen. Multi-parametric immunophenotyping was performedon 4×10⁶ total cells/condition. Cells were stained at a density of 1×10⁶cells/100 μl PBS for 30 minutes on ice using antibody and reagentconcentrations recommended by the manufacturer, washed, and acquiredusing a modified LSRII (BD Immunocytometry systems) equipped with Blue(488 nm) Violet (405 nm), Green (532), and Red (633 nm) lasers andappropriate filter sets for the detection and separation of the aboveantibody combinations. A minimum of 100,000 CD3+ cells were acquired foreach stain. For functional assays, cells were washed, stained forsurface markers, and acquired as above; a minimum of 50,000 CD3+ eventswere collected for each staining condition. Compensation values wereestablished using single antibody stains and were calculated and appliedautomatically by the instrument. Data were analyzed using FlowJosoftware (Version 8.8.4, Treestar).

Xenogeneic Lymphoma Models

To assess the persistence and antitumor effect of different CAR (UW022,UW026 and FMC63) modified CD3⁺ lymphocytes in vivo, we used a severecombined immunodeficient (SCID)-lymphoma human xenograft model. AdvancedSevere Immuno Deficiency (ASID) mouse(NOD.Cg-Prkdcsc^(scid)Il2rg^(tm1Wjl)/YckNarl) experiments were performedin accordance with the Kaohsiung Veterans General Hospital InstitutionalAnimal Care and Use Committee.

The first step of experiment is to evaluate Raji/Luc⁺ cells engraftment.Therefore, ASID mice (8-10-week old; Laboratory Animal Center, Taiwan;5×10⁵ per mouse) were injected intraperitoneally (i.p.) with Raji/Luc⁺(5×10⁵ cells per mouse) at Day 0. Tumor engraftment was measured usingthe in vivo imaging system as following described. In brief, mice wereinjected intraperitoneally (i.p.) with D-luciferin potassium salt (3mg/mouse; Perkin Elmer; Waltham, Mass.), and analyzed using the IVISSpectrum (Caliper Life Sciences, Hopkinton, Mass.). Photons emitted fromluciferase-expression cells were quantified using the Living Image 3.0software program. Mice were euthanized when bioluminescence reached1×10¹⁰ photons/second, or earlier if they showed physical signswarranting euthanasia.

To further determine the antitumor effect of different anti-CD19 CAR(UW022, UW026, and FMC63) modified CD3⁺ lymphocytes, the lymphocytesthereof are expanded. After, T cells transduced with anti-CD19 CAR(UW022, UW026, and FMC63) and mock-transduced T cells were resuspended(RPMI-1640 plus 10% FBS) and expanded for 8 days, and then injected i.p.(1×10⁷ cells per mouse) 7 days (at Day 7) after Raji-Luc injection. As acontrol, a group of mice received tissue culture medium instead of Tcells.

Statistical Analysis

Student's t-test was used to determine the statistical significance ofdifferences between samples, and P<0.05 was accepted as indicating asignificant difference. For the bioluminescence experiments, intensitysignals were summarized using mean±s.d. at baseline and multiplesubsequent time points for each group of mice.

EXAMPLES

The following examples are offered by way of example and are notintended to limit the scope of the invention in any manner.

Example 1 Various Artificial CAR19 T Cells Demonstrate Similar ExpansionProfile

Three different artificial CAR19 expression vector, i.e. UW022, UW024and UW026, are constructed as the previous description. Further, theartificial CAR19 generated by the UW022, UW024 and UW026 generallyshares the similar composition that including a scFv in the CD19antigen-binding fragment, a CD8α transmembrane domain and a signalingdomain is consisted of a CD3 zeta chain (i.e., ITAM motif) and a4-1BB/CD137 (i.e., CM motif.) Moreover, the amino acid combination of aheavy chain variable region and a light chain variable region of thescFv of the CAR19 in the UW022, UW024 and UW026 are SEQ ID NO: 8 withSEQ ID NO: 7, the SEQ ID NO: 10 with SEQ ID NO: 9, and the SEQ ID NO: 12with SEQ ID NO: 11 respectively. The nucleic acid sequence combinationof a heavy chain variable region and a light chain variable region ofthe scFv of the CAR19 in the UW022, UW024 and UW026 are SEQ ID NO: 2with SEQ ID NO: 1, SEQ ID NO: 4 with SEQ ID NO: 3, and SEQ ID NO: 6 withSEQ ID NO: 5. In this experiment, another artificial CAR19 (i.e., FMC63)is used as the comparison. Through the GFP of the expression vector andthe anti-Fab antibody, the transfection efficiency and the CAR19 proteinexpression of UW022, UW024 and UW026 can be detected. According to thequantitative results of GFP-positive CD3⁺ lymphocytes (i.e., T cells) inFIGS. 2A and 2B, UW022-transfected T cells show the highest amountGFP-positive cells (29.71%) than others (UW024: 9.67%; UW026: 12.86%; orFMC63: 6.32%). That is the expression vector of UW022 shows the highesttransfection efficiency compared to others modified CAR19 vectors.According to the quantitative results of Fab expression on primary humanCD3⁺ lymphocytes in FIGS. 2A and 2B, UW022 (21.6%), UW024 (9.77%) andUW026 (10.1%) all show that able to express the higher artificial CAR19protein on CD3⁺ lymphocytes surface than FMC63 (2.22%). Furthermore, itis worth to note that UW022 shows significantly higher efficiency to betransfected into the CD3⁺ lymphocytes and then induces the CD3⁺lymphocytes to express higher amount of artificial CAR19 protein thanFMC62 (as FIG. 2B shows). Taken together, the artificial CAR19expression vector of UW022 shows both the high transfection efficiencyand high expression efficiency compared to UW024, UW026 and FMC63.Although both UW024 and UW026 show the less ability of the transfectionand the expression, both of them still demonstrate the higher alibiesthereof compared to the FMC63. Additionally, UW024 and UW026 demonstratethe similar pattern (i.e., transfection efficiency and expressionefficiency) with each other. To further determinate whether abio-activity (i.e., proliferation ability/rate) of the modified T cellswith UW022, UW024, UW026 or FMC63 (herein after “UW022-T cells, UW024-Tcells, UW026-T cells or FMC63-T cells”) is similar to or correlated withthe above results. In other words, whether the UW022-T cells show thehighest proliferation ability among the UW024-T cells, UW026-T cells orFMC63-T cells? The GFP and Fab double positive UW022-T cells, UW024-Tcells, UW026-T cells or FMC63-T cells are purified and further culturedfor the designed time. The proliferation ability of each group cells isexamed by cell counting at day 2, 5 and 8 after culture. According tothe FIG. 2C, cells growth rate of the UW022-T cells, UW024-T cells,UW026-T cells or FMC63-T cells are similar, therefore the proliferationability thereof are similar.

Example 2 UW022-T Cells Demonstrate Highest Cytotoxicity Ability to CD19Positive Tumor Cells

To evaluate cytotoxicity ability of each modified CAR19 T cells (i.e.,UW022-T cells, UW024-T cells, UW026-T cells or FMC63-T cells) to CD19positive tumors, the certain modified CAR19 T cells are co-cultured withRS4;11 cells and Raji cells for indicating time and then analyzes anumber of the CD19 positive cells through the flow cytometry afterco-culture experiment. As the results of FIGS. 3A and 3B, although eachof modified CAR19 T cells demonstrates that are capable of killing bothCD19 positive tumor cells, the UW022-T cells show the highestcytotoxicity ability (only 4.01% of RS4;11 cells remain; only 0.41% ofRaji cells remain) compared to others modified CAR19 T cells. Regardingthe results of the FIGS. 3A and 3C, although the UW024-T cells (41.70%of RS4;11 cells remain), UW026-T cells (38.50% of RS4;11 cells remain)and FMC63-T cells (45.10% of RS4;11 cells remain) show less cytotoxicityability than UW022-T cells, both UW024-T cells and UW026-T cells stillhave higher ability than the FMC63-T cells to the RS4;11 tumor cells.Regarding the results of the FIGS. 3B and 3D, all of the CAR19 T cellsshow significant cytotoxicity ability to the Raji cells, however, theability of the UW022-T cells (0.41% of Raji cells remain), the UW024-Tcells (2.60% of Raji cells remain) and the UW026-T cells (5.16% of Rajicells remain) are still higher than the FMC63-T cells (7.42% of Rajicells remain).

For further confirming the results of cytotoxicity ability of FIG. 3A to3D, the quantity of IFN-γ that secreted by the modified CAR19 T cells inthe culture medium is analyzed through the enzyme-linked immunosorbentassay (the “ELISA assay”). According to the result of FIGS. 3E and 3F,the UW022-T cells is capable of secreting the highest amount of IFN-γ tothe RS4;11 cells or the Raji cells compared with others modified CAR19 Tcells. Furthermore, it is worth to note that UW022-T cells are capableof secreting significant higher amount of IFN-γ compared with the Mocktreatment to the RS4;11 cells or the Raji cells. Therefore, the modifiedCAR19 T cells indeed have the cytotoxicity ability to the CD19 positivetumor cells (e.g., the RS4;11 cells and the Raji cells). Furthermore,the UW022-T cells demonstrate the significant cytotoxicity abilitycompared with others CAR19 T cells.

To further evaluate the limitation of cytotoxicity ability of each CAR19T cells, we culture the certain CAR19 T cells with the tumor cells inthe different ratio (e.g., the CAR19 T cells versus the tumor cells in1:1, 2:1, 5:1 or 10:1). Furthermore, the tumor cells used herein includetwo CD19-positive tumors (i.e., RS4;11 and Raji) and one CD19-negativecell (i.e., K562). Regarding the results of K562 cells, all fourmodified CAR19 T cells show the similar cytotoxicity ability, and theability correlates with the CAR19 T cell number. However, increasing theCAR19 T cells only slightly increases the ability (i.e., donor 1: formabout 7% to about 17% and donor 2: from about 24% to about 40%).

Regarding the results of RS4;11 and Raji cells, all four modified CAR19T cells show the similar cytotoxicity ability that the abilitycorrelates with the CAR19 T cell number. According to the results fromTable 1 and FIG. 4A, the UW022-T cells show the highest cytotoxicityamong all CAR19 T cells to RS4;11 or Raji cells. However, increasing thecell number of the UW022-T cells only slightly enhances the cytotoxiceffect in both CD19-positive tumor groups. In another side, increasingothers CAR19 T cells numbers may enhance the cytotoxicity to the tumor.For example, doubling the cells numbers of the UW026-T cells increasesthe cytotoxicity from 19.79% to 53.30% in the donor 1 group. Yet, onlythe UW022-T cells can result in the highest cytotoxicity in variousratio to the different tumor cells.

TABLE 1 the cytotoxicity of different ratio of CAR19 T cells to tumorcells RS4; 11 Raji K562 Ratio (CAR19 T cell: Tumor cell) Groups 5:1 10:11:1 2:1 1:1 2:1 Donor 1 Mock 4.22% 7.39% 5.03% 22.94% 9.84% 10.78% FMC63T cells 45.78% 55.80% 4.17% 25.29% 6.33% 10.30% UW022-T cells 52.90%60.95% 43.43% 51.99% 6.20% 22.85% UW024-T cells 42.74% 54.88% 29.39%44.99% 4.52% 12.73% UW026-T cells 19.79% 53.30% 23.91% 41.30% 7.51%15.49% Donor 2 Mock 20.05% 42.22% 20.19% 39.42% 28.50% 37.03% FMC63 Tcells 65.64% 77.08% 46.37% 59.85% 18.54% 38.25% UW022-T cells 70.66%76.88% 50.67% 58.66% 22.49% 39.33% UW024-T cells 41.44% 63.10% 33.98%53.29% 20.93% 38.60% UW026-T cells 57.93% 74.78% 27.92% 53.68% 22.96%43.55%

We also used the flow cytometry assay to confirm the previous result.The previous treatment cells are further performed the double stainingby propidium iodide (PI) and calcein-AM, and the staining cells areanalysis through a flow cytometer. The PI and calcein-AM dyes were usedto stain viable and dead cells, respectively. The calcein-AM dye onlystains viable cells, and the PI only stains dead cells (i.e., necrosiscells). According to the result of FIG. 4B, the UW022-T cells induced asignificant amount of PI positive but calcein-AM negative cells than theFMC63-T cells. That is, in conclusion, the UW022-T cells can effectivelyinduce the tumor cells necrosis, which more specifically is the CD19positive tumor cells.

Example 3 CAR19 T Cells Enhance the Cytotoxicity Ability to CD19Positive Tumor Cells In Vivo

To evaluate the persistence of our modified T cells in vivo, we used anASID mouse lymphoma xenograft and an extensively validatedbioluminescence imaging system (e.g., IVIS system). The ASID mouselymphoma xenograft experiment design is as FIG. 5A shows. We began byevaluating the tumor engraftment after i.p. inoculation of Raji cellslabeled with FFLuc at Day 0. After inoculating the FFLuc-labeled Rajicells, the growth of the FFLuc-labeled Raji cells in the ASID mouse weremonitored by the IVIS system at Day 6, 14, 21, 28 and 34. We found thatafter infusion, Raji (5×10⁵ cells) had engrafted diffusely in lymphnodes and intraperitoneal (as the none-treatment group shows in FIG.5B). After defining the timing and sites of tumor engraftment, weassessed T-cell trafficking to the tumor and T cell persistence in vivo.Control mock and three CAR19 T cells (FMC63-T, UW022-T and UW026-T) wereinfused (1×10⁷ cells/mouse) in mice after engrafting with labeled Rajicells. Specifically, the first step of the experiment is to induce thetumor model on the ASID mice through i.p. inoculation of Raji cellslabeled with FFLuc into ASID mice at Day 0. After six days of i.p.inoculation (i.e., at Day 6), we use IVIS system to confirm that theinoculated FFLuc labeled Raji cells induce the solid tumor on the micegrew to the size that we expect. After confirming all the mice have thesimilar size of the solid tumor, we further treats the mice with controlmock T cells and three CAR19 T cells (FMC63-T, UW022-T and UW026-T) atDay 7. We also use IVIS system to image the tumor growth every sevendays (i.e., at Day 14, 21, 28 and 34) after treating mice with controlmock T cells and three CAR19 T cells. Briefly, we only respectivelyinject the FFLuc labeled Raji cells and the various T cells into themice once.

FIGS. 5B and 5C illustrate the development of Raji-engrafted tumor cellsafter infusing with various T cells. In one aspect, Raji bioluminescenceis easily detectable when Raji was engrafted after 6 days (as the imagein the Day 6 in the FIG. 5B shows). In another aspect, all T cellsinfused groups (e.g., the control mock T cell group and three CAR19 Tcells group (e.g., FMC63-T, UW022-T and UW026-T)) show the less Rajibioluminescence signal than none treatment group. Yet, two of UW026-Tcells treatment mice demonstrate the contrary results (i.e., abundantRaji cells development). As FIGS. 5B and 5C show, mock-T cells alsocapable of decreasing and delaying the tumor development compared withnone treatment group. However, only FMC63-T and UW022-T cellssignificantly inhibit the solid tumor development. Importantly, UW022-Tcells show the highest efficiency to inhibit the Raji cells developmentcompared with FMC63-T cells. After engrafting UW022-T cells, thebioluminescence signal of the tumor cells in the mice of the UW022-Ttreatment group barely increases in the following detection comparedwith others treatment group or none treatment group. On other words, thetumor size on the mice of the UW022-T treatment group does notsignificantly increase compared with others groups. In conclusion,UW022-T cells also demonstrate the higher cytotoxicity than FMC63-Tcells in vivo.

Although the present disclosure and its advantages have been describedin detail, it should be understood that various changes, substitutionsand alterations can be made herein without departing from the spirit andscope of the disclosure as defined by the appended claims. For example,many of the processes discussed above can be implemented in differentmethodologies and replaced by other processes, or a combination thereof.

Moreover, the scope of the present application is not intended to belimited to the particular embodiments of the process, manufacture,composition, methods and steps described in the specification. As one ofordinary skill in the art will readily appreciate from the disclosure ofthe present disclosure, processes, manufacture, compositions, methods,or steps, presently existing or later to be developed, that performsubstantially the same function or achieve substantially the same resultas the corresponding embodiments described herein may be utilizedaccording to the present disclosure. Accordingly, the appended claimsare intended to include within their scope such processes, manufacture,compositions, methods, or steps.

What is claimed is:
 1. An artificial chimeric antigen receptor (CAR),comprising: a CD19 antigen-binding fragment, wherein the CD19antigen-binding fragment is a single-chain variable fragment (scFv), andthe scFv comprises a heavy chain variable domain, a light chain variabledomain and a linker, wherein the heavy chain variable domain comprises afirst amino acid sequence selected from SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO: 12 or any combination thereof, and the light chain variabledomain comprises a second amino acid sequence selected from SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11 or any combination thereof; atransmembrane domain comprising a transmembrane domain of CD28, IgG1,CD4, CD8α or any combination thereof; and a signaling domain comprisingan immunoreceptor tyrosine-based activation motif (ITAM), aco-stimulatory molecule (CM) or combination thereof.
 2. The artificialCAR according to claim 1, wherein the transmembrane domain is thetransmembrane domain of CD8a.
 3. The artificial CAR according to claim1, wherein a number of the CM is at least two.
 4. The artificial CARaccording to claim 1, wherein the ITAM comprises CD3 zeta.
 5. Theartificial CAR according to claim 1, wherein the CM comprises a CM ofCD27, CD28, CD30, 4-1BB/CD137, OX40, herpesvirus entry mediator (HVEM)or any combination thereof.
 6. The artificial CAR according to claim 1wherein the CM comprises a CM of 4-1BB/CD137.
 7. An expression vector ofartificial CAR, comprising: a CD19 antigen-binding fragment sequencecomprising a nucleic acid sequence of a heavy chain variable domain, alight chain variable domain and a linker, wherein the nucleic acidsequence of the heavy chain variable domain selected from SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6 or any combination thereof and the nucleicacid sequence of the light chain variable domain selected from: SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or any combination thereof; atransmembrane domain sequence comprising a nucleic acid sequence of atransmembrane of CD28, IgG1, CD4, CD8α or any combination thereof; and asignaling domain sequence comprising a nucleic acid sequence of a ITAMsequence, a CM sequence or combination thereof.
 8. The expression vectoraccording to claim 7, wherein the transmembrane domain sequence is thetransmembrane sequence of CD8a.
 9. The expression vector according toclaim 7, wherein a number of the at least one CM is at least two. 10.The expression vector according to claim 7, wherein the at least oneITAM comprises CD3 zeta.
 11. The expression vector according to claim 7,wherein the sequence of the at least one CM comprises a CM sequence ofCD27, CD28, CD30, 4-1BB/CD137, OX40, HVEM or any combination thereof.12. The expression vector according to claim 11 wherein the at least oneCM sequence comprises the CM sequence of 4-1BB/CD137.
 13. Apharmaceutical composition comprising a population of a modified cell,comprising: the artificial CAR as according to claim 1, the expressionvector of artificial CAR as according to claim 7 or any combinationthereof.
 14. The pharmaceutical composition according to claim 13,wherein the modified cell is a mammalian cell.
 15. The pharmaceuticalcomposition according to claim 13, wherein the modified cell is amammalian lymphocyte.
 16. The pharmaceutical composition according toclaim 13, wherein the modified cell is a T cell or a NK cell.
 17. Amethod for treating a mammal having a disease, a disorder or a conditionassociated with an elevated expression of CD19 antigen, comprising: (a)isolating a peripheral blood from a mammalian donor; (b) purifying aplurality of lymphocytes from the peripheral blood; (c) generating apharmaceutical composition as according to claim 13 by the plurality oflymphocytes; (d) treating a mammalian recipient with a chemotherapeuticagent; and (e) administrating the pharmaceutical composition to the atmammalian recipient.
 18. The method according to claim 17, wherein the(c) further comprises amplifying the pharmaceutical composition.
 19. Themethod according to claim 17, wherein the pharmaceutical composition isautologous to the mammalian donor and the mammalian recipient.
 20. Themethod according to claim 17, wherein the pharmaceutical composition isallologous to the mammalian donor and the mammalian recipient.
 21. Themethod according to claim 17, wherein the lymphocyte is a T cell or a NKcell.